1. Field of the Invention
The present invention relates to a transparent and stable aqueous solution of triglyceride substrate for a determination of lipase, a method for a determination of lipase activity in a sample, employing the aqueous solution, and a process for the manufacture of a lyophilized product capable of forming the aqueous solution. Further, the present invention also relates to a process for the manufacture of a transparent and stable aqueous solution of triglyceride.
2. Description of the Related Art
The enzyme lipase is naturally widely present in, for example, animals or plants, and the activity thereof is determined for various purposes; i.e., from a clinical point of view, it is particularly important for an early detection of diseases in pancreas, for example, acute pancreatitis, pancreas cancer, or the like.
Various methods for the determination of lipase are known, but the following two methods are most widely employed in the clinical field:
1. Nephelometry PA1 2. Method Using Synthesized Substrate Other Than Triglyceride
This method comprises carrying out an enzymatic reaction while using a suspension of triglyceride (typically, olive oil) or glycerol ester of a higher fatty acid as a substrate, and then measuring a reduction of turbidity of the suspension by an absorbance thereof. But this method has the following disadvantages:
a) The lipase activity does not always coincide with the reduction of turbidity. For example, in emulsified samples, some samples show an increased absorbance.
b) The lipase activity is calculated from the measurement of the turbidity reduction. This is an indirect determination of the enzymatic activity.
c) When the lipase activity is low, the sensitivity and reproducibility are poor.
In this method, esters of fatty acid derivatives or alcohol derivatives, for example, monoglyceride, 1,2-diglyceride, p-nitrophenol laurate, dimethylcaprol tributyrate, .alpha.-naphthyl palmirate and the like are employed as a substrate. But, when the synthetic substrate is insoluble in water, this method encounters not only the disadvantages mentioned in the above item 1, but also a problem of a poor substrate-specificity such that the synthetic substrate may be hydrolyzed by esterases other than lipase. Although a water-soluble substrate can remedy the disadvantages mentioned in the above item 1, this method still has the problem of the substrate-specificity such that the substrate may be hydrolyzed by esterases other than lipase.
Under these circumstances, the inventors of the present invention carried out various research protects to develop a method for a determination of lipase, taking into account the following points:
a) Because lipase is the enzyme for hydrolyzing a triglyceride having long-chain fatty acids, triglyceride is used as the substrate.
b) Instead of measuring the reduction of the turbidity (substrate), the fatty acid, monoglyceride or glycerol formed from hydrolyzation of triglyceride with lipase is directly measured.
c) The method is simple and can be applied for a clinical examination.
Japanese Examined Patent Publication (Kokoku) No. 59-39168 (Japanese Patent No. 1271605) discloses a process for the manufacture of a transparent solubilized aqueous solution of a water-insoluble material. This process comprises the steps of adding the water-insoluble material into an aqueous solution containing a nonionic surfactant, heating the whole, while stirring, to a temperature higher than a cloud point of the surfactant solution, and then cooling, while stirring, to a temperature of the cloud point or less to form a transparent solution. According to this Japanese Publication 59-39168, the aqueous solution thus obtained is very stable, and turbidity does not result from a change of the pH or a decrease of the concentration by dilution with water or buffer. This Japanese publication 59-39168 also teaches that the process is very useful for the manufacture of a stable standard solution of triolein, and that the transparent aqueous solution of triolein makes it possible to simply and accurately measure the lipase activity by a photometer. Although this Japanese Publication 59-39168 contains a relatively general description, it does not describe a concrete means of actually realizing a method for the determination of lipase activity in a sample.
In connection with the transparent solubilized aqueous solution of the water-insoluble material, the inventors of the present invention found another process for the manufacture thereof, as disclosed in Japanese Application No. 61-234520 filed on Oct. 3, 1986 (published on Apr. 21, 1988 as Unexamined Publication (Kokai) No. 63-91136).
Further, the inventors found that the transparent aqueous solution disclosed in Japanese Publication 59-39168 and Japanese Application 61-234520 can be employed as a substrate solution in the determination of lipase activity.
In addition, the inventors found that this transparent aqueous solution can be stored for a long term by a lyophilization thereof without a denaturation.